High speed sample workflow for lc-ms based hba1c measurement
A sample, whole blood sample technology, applied in the field of clinical diagnostic systems, can solve the problems of time-consuming, limited high-throughput measurement of patient samples, etc.
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[0059] A first aspect of the present invention relates to a method for determining glycated hemoglobin A (HbA1c) in a sample, comprising:
[0060] (a) providing a sample comprising hemoglobin A (HbA) molecules,
[0061] (b) subjecting the sample from step (a) to a partial proteolytic digestion step in which an N-terminal fragment of the hemoglobin Aβ chain molecule is produced,
[0062] (c) optionally enriching the N-terminal fragment of the hemoglobin Aβ chain molecule produced in step (b), and
[0063] (d) Analysis of the sample comprising the N-terminal fragment of the hemoglobin Aβ chain molecule by mass spectrometry (MS), wherein the amount or concentration of HbA1c in the sample is determined.
[0064]Thus, the amount or concentration of HbA1c in a sample can be determined, in particular the relative amount of HbA1c, ie the ratio of glycated HbA1c molecules to non-glycated HbA0 molecules or to the total number of HbA molecules. When repeatedly determining the amount of...
example 1
[0186] Determination of HbA1 Peptide Fragments in Whole Blood
[0187] Dilute 100 μL of whole blood sample with 900 μL HO 2 O diluted and vortexed for 10 s until a clear solution was obtained. Then, further dilute 5 µL of the hemolyzed whole blood sample with 20 µL of HO. 5 μL of diluted hemolyzed blood sample, 15 μL intracellular protease Glu-C (2 mg / mL) and 30 μL digestion buffer (50 mM ammonium acetate pH 4.3) were added to LoBind vials (Eppendorf) and incubated at 37°C for 45 min to provide partial digestion, For example 1% to 20% of the HbA molecules present in the sample are digested. The enzymatic reaction was stopped by adding 950 μL of 0.1% (v / v) formic acid.
[0188] Then, provide several batches of 100 μL of hydrophobic magnetic beads (50 mg / mL). Mix 200 μL of digested whole blood sample with 700 μL of HO containing 0.1% (v / v) formic acid 2 O was added to each batch of beads. The mixture was shaken at 37 °C and 1200 rpm for 5 min.
[0189] Next, the beads wer...
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